Apoptosis, also referred to as programmed cell death, is a process in which a cell dies by activation of an intrinsic genetic program. Apoptosis plays an important role in the development and aging processes in the central nervous system (CNS). Abnormality in apoptosis has been linked to pathogenesis of neurodegenerative diseases and implicated in neuropsychiatric disorders. We have used primary cultures of rat CNS neurons and neurally related cell lines as a model to study molecular mechanisms underlying neuronal apoptosis. We found that glyceraldehyde-3-phospahate dehydrogenase (GAPDH), a glycolytic enzyme with multiple non-glycolytic functions, is over- expressed during apoptosis induced by aging of rat cerebellar granule cells in culture. Antisense oligonucleotides to GAPDH block GAPDH over-expression and effectively delay age-induced apoptosis of these cerebellar neurons. These results provide the first evidence for a role of GAPDH in neuronal apoptosis. By the same criteria, we found that GAPDH is involved in age-induced apoptosis of rat cerebral cortical neurons and apoptosis of cerebellar granule cells induced by extracellular potassium deprivation and exposure to cytosine arabinoside (AraC). In the case of AraC-induced apoptosis, we found that the cell death occurs within a narrow frame of approximately 40 hours after plating and is robustly protected by neurotrophins, BDNF and NT 4/ 5 but not NT-3. The apoptosis is also associated with an increased expression of two death genes, p53 and Bax, preceding the over-expression of GAPDH. An antisense oligonucleotide to p53 reduces GAPDH induction and protects against AraC-induced apoptosis, suggesting that GAPDH over-expression depends on p53 expression. Moreover, tranfection of the p53 gene into PC-12 pheochromocytoma cells induces GAPDH overexpression and causes ultimate cell death, suggesting that GAPDH is a novel target gene regulated by p53. AraC-induced GAPDH over-expression is predominantly accumulated in the nucleus assessed by subcellular fractionation study and electromicroscopic immunohistochemistry. Translocation of GAPDH to the nucleus occurs parallel with a loss of GAPDH glycolytic and uracil glycosylase activities, suggesting an alteration in the structure and function of nuclear GAPDH. In support of this possibility, we found that nitric oxide-catalyzed NAD labeling of nuclear GAPDH is greatly diminished after AraC treatment. We also employed the technique of 2-dimensional gel electrophoresis to demonstrate that at least 6 GAPDH isoforms can be detected in the nucleus of cerebellar granule cells. These nuclear isoforms differ in their abundance and are translocated to the nucleus according to a distinct time-course following AraC treatment. Evidence is also available that nuclear GAPDH accumulation precedes activation of caspase-3 and cleavage of its nuclear substrate lamin B. Recently, GAPDH has been shown to bind specifically to gene products of degenerative diseases such as Alzheimer's disease, Huntington's disease, DRPLA and spinocerebellar ataxia. Our results suggest that such specific binding could be the results of over-expression of GAPDH in CNS neurons, which in turn induces neuronal apoptosis and ultimate neurodegeneration. Moreover, the ability of GAPDH antisense oligonucleotides to rescue neurons from undergoing apoptotic cell death also suggests that such oligonucleotides may be useful for treating neurodegenerative diseases. A recent report also demonstrates that GAPDH is a putative target of deprenyl, an anti-apoptotic agent under clinical trials for Alzheimer's disease and Parkinsonism. Thus, GAPDH may be used as a therapeutic target for novel drug development. We also found that amlodipine, a calcium channel blocker with anti-oxidant activity, is and effective neuroprotectant against age-induced apoptosis of cerebellar granule cells. This property would be related to amlodipine's ability to improve cognitive function in animal behavioral studies. In this context, we also found that ONO-1603, a potential anti-dementia drug, protects against age-induced apoptotic death of cerebral cortical neurons. Additionally, ONO-1603 and tetrahydroaminoacridine, a FDA-approved anti-dementia drug, protects cerebellar neurons against low KCl- induced apoptosis. Thus, apoptotic death of CNS neurons in culture could be a valuable tool for screening more effective drugs in the treatment of dementia associated with Alzheimer's disease and AIDS. In an attempt to further elucidate molecular mechanisms underlying apoptosis, we have used cerebellar granule cells treated with AIDS-related neurotoxins, such as 3-OH- kynurenine (3-HK) and quinolinic acid, which are tryptophan metabolites and whose levels are elevated in the brain of AIDS patients. We found that 3-HK induces a robust dose-dependent apoptotic response in the range of 50-1000 micro-M, while quinolinic acid is ineffective. The 3-HK neurotoxicity is potentiated by superoxide dismutase (SOD) and a cell permeable SOD mimetic, MnTBAP. Both 3-HK-induced and SOD-potentiated neurotoxicities are blocked by catalase, suggestive of hydrogen peroxide formation. We also found that 3-HK induces apoptotic death of PC-12 pheochromocytoma cells and hypothalamic GT1-7 cells. In both cell types, 3-HK-induced apoptosis is robustly protected by dantrolene, a drug that inhibits Ca-2+ efflux from the endoplasmic reticulum to the mitochondria. Moreover, overexpression of Bcl-2, an anti-apoptotic gene product, in GT1-7 cells arrests 3-HK-induced neurotoxicity. Thus, pharmacological manipulation with dantrolene and/or gene therapy with Bcl-2 are potentially useful for the treatment of 3-HK-related neurodegeneration.